O-linked glycosylation is prominent in all domains of life, and O-glycoproteins participate in diverse cellular functions including signaling and cell communication, cell adhesion, immune surveillance and host-pathogen interactions, inflammation, and endocytosis. As a consequence, abnormal O-glycosylation is implicated in a number of human diseases including, cancer, Congenital Disorders of Glycosylation, and autoimmune diseases such as Tn syndrome and IgA nephropathy. In particular, the tumor associated O-glycans, Tn and its derivative sialyl Tn (STn), appear in the majority of cancers and are indicative of metastatic potential and poor prognosis. Tn-glycoproteins are likely to have altered function as a result of their abnormal O-glycosylation, and such outcomes may be important in promoting tumor development and metastasis. To address this, the Tn- glycoproteome of tumors must first be defined. Colorectal cancer (CRC) is the second leading cause of cancer- related deaths in the US, partly due to the lack of early detection methods. Aim 1 will define the Tn-glycoproteome in human colorectal tumor specimens, using a robust proteomics methodology for identifying Tn-glycopeptides from cell lysates. We expect to identify O-glycoprotein membrane proteins including signaling receptors, adhesion receptors, and signaling ligands that may be useful targets for therapeutic intervention, and soluble Tn-glycoproteins that are potentially valuable biomarkers for early diagnosis. In disease models Tn expression is often a direct result of reduced or loss of T-synthase activity, which appends a galactose residue to the Tn antigen. T-synthase activity requires a specific protein chaperone, termed Cosmc, and expression of both T-synthase and Cosmc are essential for normal O-glycosylation. Reduced expression of Cosmc has been identified as the cause of Tn expression in human and mouse tumor cell lines and in Tn syndrome patients. Although Cosmc has emerged as a key regulator of Tn expression in vivo, a biophysical description of Cosmc/T-synthase interactions is lacking. Aim 2 will (A) determine mechanisms of Tn expression by characterizing T- synthase and Cosmc interactions using biophysical tools, and (B) identify alternative chaperones using high- throughput screening. Determining the molecular mechanisms of Cosmc/T-synthase interactions will directly suggest other strategies that mimic the in vivo activation pathway, including molecular chaperones. Alternative molecular chaperones such as pharmacological chaperones and antibodies are a new class of valuable therapeutics that could be useful in the treatment of Tn diseases, including cancer, IgA nephropathy, and Tn syndrome.